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[
J Cell Biol,
2003]
During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.
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[
PLoS One,
2012]
As part of a multi-subunit ring complex, -tubulin has been shown to promote microtubule nucleation both in vitro and in vivo, and the structural properties of the complex suggest that it also seals the minus ends of the polymers with a conical cap. Cells depleted of -tubulin, however, still display many microtubules that participate in mitotic spindle assembly, suggesting that -tubulin is not absolutely required for microtubule nucleation in vivo, and raising questions about the function of the minus end cap. Here, we assessed the role of -tubulin in centrosomal microtubule organisation using three-dimensional reconstructions of -tubulin-depleted C. elegans embryos. We found that microtubule minus-end capping and the PCM component SPD-5 are both essential for the proper placement of microtubules in the centrosome. Our results further suggest that -tubulin and SPD-5 limit microtubule polymerization within the centrosome core, and we propose a model for how abnormal microtubule organization at the centrosome could indirectly affect centriole structure and daughter centriole replication.
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[
Methods Mol Biol,
2009]
In this chapter we describe the preparation of early mitotic C. elegans embryos for the tomographic reconstruction of end-morphologies of spindle microtubules. Early embryos are prepared by high-pressure freezing and freeze-substitution for thin-layer embedding in Epon/Araldite. We further describe data acquisition, tomographic reconstruction, and 3-D modeling of microtubules in serially sectioned mitotic spindles. The presented techniques are applicable to other model systems.
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[
J Microsc,
2003]
Caenorhabditis elegans is one of the most important genetic systems used in current biological research. Increasingly, these genetics-based research projects are including ultrastructural analyses in their attempts to understand the molecular basis for cell function. Here, we present and review state-of-the-art methods for both ultrastructural analysis and immunogold localization in C. elegans. For the initial cryofixation, high-pressure freezing is the method of choice, and in this article we describe two different strategies to prepare these nematode worms for rapid freezing. The first method takes advantage of transparent, porous cellulose capillary tubes to contain the worms, and the second packs the worms in E. coli and/or yeast paste prior to freezing. The latter method facilitates embedding of C. elegans in a thin layer of resin so individual worms can be staged, selected and precisely orientated for serial sectioning followed by immunolabelling or electron tomography.
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[
Curr Biol,
2006]
Katanin is a heterodimer that exhibits ATP-dependent microtubule-severing activity in vitro . In Xenopus egg extracts, katanin activity correlates with the addition of cyclin B/cdc2, suggesting a role for microtubule severing in the disassembly of long interphase microtubules as the cell prepares for mitosis . However, studies from plant cells , cultured neurons , and nematode embryos suggest that katanin could be required for the organization or postnucleation processing of microtubules, rather than the dissolution of microtubule structures. Here we reexamine katanin''s role by studying acentrosomal female meiotic spindles in C. elegans embryos. In mutant embryos lacking katanin, microtubules form around meiotic chromatin but do not organize into bipolar spindles . By using electron tomography, we found that katanin converts long microtubule polymers into shorter microtubule fragments near meiotic chromatin. We further show that turning on katanin during mitosis also creates a large pool of short microtubules near the centrosome. Furthermore, the identification of katanin-dependent microtubule lattice defects supports a mechanism involving an initial perforation of the protofilament wall. Taken together, our data suggest that katanin is used during meiotic spindle assembly to increase polymer number from a relatively inefficient chromatin-based microtubule nucleation pathway.
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[
Methods Cell Biol,
2007]
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[
Dev Cell,
2005]
In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.
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[
Methods Cell Biol,
2012]
This chapter is an update of the previously published book chapter "Correlative Light and Electron Microscopy of Early C. elegans Embryos in Mitosis" (Muller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007). Here, we have adapted and improved the protocol for the isolated meiotic embryos, which was necessary to meet the specific challenges a researcher faces while investigating the development of very early Caenorhabditis elegans embryos ex-utero. Due to the incompleteness of the eggshell assembly, the meiotic embryo is very fragile and much more susceptible to changes in the environmental conditions than the mitotic ones. To avoid phototoxicity associated with wide-field UV illumination, we stage the meiotic embryos primarily using transmitted visible light. Throughout the staging and high-pressure freezing, we incubate samples in an isotonic embryo buffer. The ex-utero approach allows precise tracking of the developmental events in isolated meiotic embryos, thus facilitating the comparison of structural features between wild-type and mutant or RNAi-treated samples.
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[
Methods Cell Biol,
2019]
We describe a routine method to locate cells of appropriate meiotic stages in the gonad of Caenorhabditis elegans males prior to 3D reconstruction of meiotic spindles by electron tomography. For this, serial semi-thick (300nm) sections of whole worms are pre-screened and recorded at low magnification by transmission electron microscopy. Cells of interest are identified in aligned image stacks showing the entire proximal region of male gonads at low magnification. Tilt series of selected cells are then recorded at higher magnification to reconstruct meiotic spindles of selected cells in 3D. Our approach allows a routine staging of spermatocytes without the use of anesthetics or the application of physical immobilization of worms. We also describe a modification of a previously published protocol (Muller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007) by using polyvinylpyrrolidone (PVP) instead of bovine serum albumin (BSA) as a "filler" for specimen loading in high-pressure freezing.
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de Bono, Mario, Amin-Wetzel, Niko, Sengupta, Piali, Philbrook, Alison, Kazatskaya, Anna, Yuan, Lisa
[
MicroPubl Biol,
2020]
A subset of sensory neurons in C. elegans contains compartmentalized sensory structures termed cilia at their distal dendritic ends (Ward et al. 1975; Perkins et al. 1986; Doroquez et al. 2014). Cilia present on different sensory neuron types are specialized both in morphology and function, and are generated and maintained via shared and cell-specific molecules and mechanisms (Perkins et al. 1986; Evans et al. 2006; Mukhopadhyay et al. 2007; Mukhopadhyay et al. 2008; Morsci and Barr 2011; Doroquez et al. 2014; Silva et al. 2017). The bilaterally symmetric pair of URX oxygen-sensing neurons in the C. elegans head (Figure 1A) is thought to be non-ciliated (Ward et al. 1975; Doroquez et al. 2014) but nevertheless exhibits intriguing morphological similarities with ciliated sensory neurons. URX dendrites extend to the nose where they terminate in large bulb-like complex structures (Ward et al. 1975; Doroquez et al. 2014; Cebul et al. 2020) (Figure 1A). These structures concentrate oxygen-sensing signaling molecules (Gross et al. 2014; Mclachlan et al. 2018) suggesting that similar to cilia, these structures are specialized for sensory functions. Microtubule growth events similar to those observed in ciliated sensory neurons were also reported at the distal dendritic regions of URX, implying the presence of a microtubule organizer such as a remodeled basal body (Harterink et al. 2018). Moreover, a subset of ciliary genes is expressed in URX (Kunitomo et al. 2005; Harterink et al. 2018; Mclachlan et al. 2018). We tested the hypothesis that URX dendrites contain cilia at their distal ends.